Drug-resistance genes have been used as a means for artificial selection of transformants in the genetic engineering field. Specifically, a desired transformant can be selected based on a drug-resistance gene whose expression confers drug resistance. Not only the drug-resistance genes but also, for example, auxotrophic genes are used as means for selecting transformants. These genes usable for selecting transformants are generally called selectable markers (selectable marker genes).
Selectable marker genes are useful for selecting transformants. However, such a selectable marker gene, depending on its kind, unfavorably exhibits adverse effects on the environment, such as biological pollution of a wild-type microorganism by the selectable marker gene, if the selectable marker gene remains in a host cell. In addition, if multiple gene manipulations are performed on the same host, the kind of selectable marker genes that can be used may be limited. For that reason, it is desired to develop a means for removing the selectable marker gene from the transformant.
The selectable marker gene is also used when a mutant is constructed by deleting a particular region from a host genome or by inserting a foreign DNA into a host cell.
For example, JP-A-2007-111015 discloses a method for deleting a particular region in host genome by using a repressor and a promoter regulated by the repressor. Specifically, the method discloses that a DNA segment (donor DNA) is integrated into a host genome by homologous recombination, and then both of the donor DNA and the particular region of the host genome are deleted from the host genome by homologous recombination between a region of the donor DNA and a region of the host genome. In this method, the donor DNA contains a selectable marker and a gene encoding a repressor, and the host genome contains a promoter under the control of the repressor and a drug-resistance gene under the control of the promoter. A transformant in which the donor DNA is incorporated into the host genome can be selected by the selectable Marker derived from the donor DNA. In addition, when the donor DNA is integrated into the host genome, the host cell develops a drug-sensitivity by repressing expression of the drug-resistance gene. After the donor DNA is removed from the host genome, the expression of the drug-resistance gene is restored to give the drug-resistant host cell. As a result, it is possible to delete the particular region of the host genome without leaving the selectable marker derived from the donor DNA in the host genome.
As explained above, the method of JP-A-2007-111015 is advantageous in that the selectable marker gene can be used effectively. However, the method requires the additional step of introducing into the host genome beforehand an expression cassette which contains the promoter under the control of the repressor and the drug-resistance gene expressed under the control of the promoter.
In cloning a gene or a DNA segment, it is very difficult to insert a gene encoding a membrane protein, a gene encoding a protein that functions as a fatal factor in a host when the protein is present in a large amount (the presence of the protein in a large amount functions as a lethal factor in a host cell), a very large-sized DNA segment, and the like into a plasmid. When a plurality of genes are involved in a particular biosynthesis or a plurality of genes constituting a subunit, it is also difficult to insert these genes and express them in a cell, simultaneously. To overcome the problem of cloning such a gene or a DNA segment, a method of using a low-copy plasmid, a method of using a bacteriophage or a cosmid generally used for preparation of a genome library as a vector, and the like have been proposed. In addition, a method of using a promoter that can strictly regulate gene expression and a method of improving chaperone function of the host Escherichia coli have also been proposed.
However, the method of improving the host often depends on the specificity of the desired product, and requires the selection and adjustment of a cloning process suitable for the specificity. In the method of using the phage or cosmid, it is also difficult to insert a plurality of genes into a plurality of gene loci of a cell.
Accordingly, there is a desire for development of a method of inserting a plurality of and relatively large-sized DNA fragments into a single cell with a simple procedure compared with the conventional method of using a phage. In recent years, DNAs obtained from environmental materials are eagerly studied by means of metagenome analysis. There is a need for a new tool and a new method of cloning genes and DNA segments that are hard to clone by conventional methods.